Interpretive Summary: Lab-based assays for detection of plant pathogens have become the cornerstone of disease management and clean stock programs. Nucleic acid-based assays using defined genome sequences are now replacing or supplementing in planta indexing as the method for diagnosis of graft-transmissible disease agents of citrus. Polymerase chain reaction (PCR) is the most sensitive procedure for pathogen detection. Quantitative real-time (q) PCR is more sensitive, faster and easier to perform than conventional PCR with reduced cycle times and no need for electrophoresis, staining or gel documentation. Furthermore, qPCR assays based on TaqMan chemistry allow for multiple target detection of different pathogens in a single-tube assay. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six pathogens including viruses, viroids, phloem-limited fastidious prokaryotes and a citrus RNA internal control. Total nucleic acid (TNA) from pathogen-infected and healthy citrus was extracted and purified using a magnetic bead-based kit and a high-throughput extraction robot. TNA samples were compared with a tissue print method and manual extraction for efficacy and suitability for use in qPCR assays. The first assay included primers and probes for “Candidatus” Liberibacter asiaticus (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal host control. TNA quality based on spectrophotometric analysis (OD 260/280) and target quantitation cycle values indicated high throughput extraction was as effective as manual extraction for pathogen detection. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, high throughput extraction and multiplex RT-qPCR assays, as described, provided a rapid, standardized and cost-effective method for routine and simultaneous diagnosis of RNA and DNA citrus pathogens from different taxa.

Technical Abstract:
A number of economically important citrus pathogens are spread by nursery propagation, arthropod vector transmission and in advertent importation and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs need to employ an economical and sensitive pathogen detection system to maintain a healthy, and robust citrus industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed in a high-throughput format for simultaneous detection of some key citrus pathogens. Automated high-throughput extraction (HTE) system comparing several bead-based commercial extraction kits were tested and compared with a tissue print method and manual extraction procedures to obtain high quality total nucleic acid (TNA) from pathogen-infected and healthy citrus trees. TNA samples were subsequently used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including viruses, viroids, phloem-limited fastidious prokaryotes and the a plant RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were used to determine sensitivity and specific detection of target templates. The first assay included primers and probes for “Candidatus” Liberibacter asiaticus (CLas), for Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and for the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal host control. TNA quality based on spectrophotometric analysis (OD 260/280) and target quantitation cycle values indicated HTE was as suitable as manual extraction for pathogen detection. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, HTE and multiplex RT-qPCR assays, herein described, provided a rapid and standardized method for diagnosis of RNA and DNA citrus pathogens from different taxa.